Insights into Bidirectional Gene Expression Control Using the

Cell Reports, Volume 25

Supplemental Information

Insights into Bidirectional Gene Expression Control Using the Canonical GAL1/GAL10 Promoter Gregory L. Elison, Yuan Xue, Ruijie Song, and Murat Acar

I.

Supplemental Figures and Legends

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Figure S1. Edited promoters in the native GAL locus behave the same as those in the ho locus. Related to Figure 1. Black arrows indicate where the promoter has been edited. Upper bars show the wild type genotype of the region being edited. Upper bars show the wild type genotype of the region being edited. Lower bars show the same region containing whatever edits, were made which are indicated by capital letters. The wild type does not have bars, as it is unedited. Bases written in a colored font are provided to indicate bases which are part of the binding site depicted in that color in Figure 1A. Panels depicting a FACS histogram and data taken from those histograms were created using 10,000 cells of the strain represented by the lower bar. Strain data error bars indicate SEM (n=2). A. Expression histogram from the wild type GAL1 promoter (strain GE1). B. Expression histogram from an edited GAL1 promoter (with edits immediately upstream of the first Gal4 binding site; strain GE210), indicating a loss of GAL1 expression. C. Expression histogram from an edited GAL10 promoter (with edits immediately upstream of the first Gal4 binding site; strain GE218), indicating a return to wild type expression. D-F. Expression histograms of strains in which the GAL genes and the PGAL1 – YFP construct in the ho locus have been swapped, with the GAL genes moved to the ho locus and the construct moved to the gal locus. D. Expression histogram of the wild type GAL1 promoter (strain GE234). E. Expression histogram from an edited GAL1 promoter (with edits immediately upstream of the first Gal4 binding site; strain GE235), indicating a loss of GAL1 expression. F. Expression histogram from an edited GAL10 promoter (with edits immediately upstream of the first Gal4 binding site; strain GE236), indicating a return to wild type expression. G. Comparison of promoter expression in the ho locus to promoter expression in the native GAL locus. Three strains are compared. WT refers to the wild type promoter driving YFP in the indicated locus. Edit1 refers to the edited promoter shown in the cartoon driving YFP in the indicated locus. This promoter is tested for both GAL1 and GAL10 expression. H. A cartoon of the promoter edit present in panels (B,C,E,F).

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A

B 100 Fraction of ON Cells (%)

90 80 70 60 50 40 30 20 10 0 0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

[galactose] (% w/v)

3

Figure S2. Growth comparison between WT strain and strain with the GAL genes moved to the ho locus, and full wild type PGAL1-YFP induction profile. Related to Figure 1. A. Growth rate measurement of the wild type strain (carrying the PGAL1 – YFP construct in the ho locus, strain GE1) and a strain in which the GAL1/GAL10 genes and the PGAL1 – YFP construct in the ho locus have been swapped, with the GAL1/GAL10 genes moved to the ho locus and the construct moved to the gal locus (strain GE229). Cells were grown in minimal media containing 0.1% galactose as the sole carbon source. Prior to OD600 measurements, cultures were grown for 22 hours in 5 ml volume in a 30C shaker-incubator (225 rpm). Then, cell densities were measured by taking aliquots from continuously growing cultures, and OD600 values were fitted to an exponential function (R2: 0.99 and 0.98). The doubling times of the two strains were calculated to be 142 min and 144 min, as shown above. B. The percentage of ON cells for the wild type strain (strain GE1) shown in Fig. 1A grown in 0.1% mannose, and varying concentrations of galactose was plotted. All data is generated from FACS histograms created using 10,000 cells. Data error bars indicate SEM (n=2).

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Figure S3. Fitness consequences of introducing edits into the GAL1/GAL10 locus. Related to Figure 2A. Three yeast strains containing edits in their endogenous GAL1/GAL10 bidirectional promoters driving the GAL1 and GAL10 genes were compared for growth rate differences to the strain containing the wild type promoter in the GAL1/GAL10 locus. The wild type strain (strain GE1) had an approximate doubling time of 100 minutes, which is normal for growth in complete synthetic media. A second strain (strain GE6) containing a deletion of the entire endogenous GAL1/GAL10 promoter (therefore preventing expression of either GAL1 or GAL10) resulted in a higher doubling time of approximately 104 minutes. A third strain (strain GE123) containing an edit on the endogenous GAL1/GAL10 promoter which eliminates GAL1 but not GAL10 expression showed the highest growth rate defect among the three strains. A fourth strain (strain GE124) containing a promoter edit which eliminates GAL10 but not GAL1 expression showed a doubling time which was approximately equivalent to the wild type strain’s doubling time. Data error bars indicate SEM (n=2).

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Figure S4. Promoter edits which disrupt GAL1 expression have little to no effect on GAL10 expression. Related to Figure 1 D,E. Black arrows indicate where the promoter has been edited. Upper bars show the wild type genotype of the region being edited. Lower bars show the same region containing whatever edits, were made which are indicated by capital letters. The wild type does not have bars, as it is unedited. Bases written in a colored font are provided to indicate bases which are part of the binding site depicted in that color in Figure 1A. Panels depicting a FACS histogram were created using 10,000 cells of the strain represented by the lower bar. Strain data error bars indicate SEM (n=2). A. GAL10 expression from the strain shown in Figure 1D (strain GE218). B. GAL10 expression from the strain shown in Figure 1E (strain GE192).

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Figure S5. Edits 5’ of the first Gal4 binding site which were unable to alter expression of GAL10. Related to Figure 2B. Black arrows indicate where the promoter has been edited. Upper bars show the wild type genotype of the region being edited. Lower bars show the same region containing whatever edits, were made which are indicated by capital letters. The wild type does not have bars, as it is unedited. Bases written in a colored font are provided to indicate bases which are part of the binding site depicted in that color in Figure 1A. Panels depicting a FACS histogram were created using 10,000 cells of the strain represented by the lower bar. Strain data error bars indicate SEM (n=2). A. Strain in which the bases TGCT were changed to AAAA (strain GE166). B. Strain in which the bases AT were changed to GG. (strain GE195)

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Figure S6. Promoter edits which disrupt GAL10 expression have little to no effect on GAL1 expression. Related to Figure 2B,C. Black arrows indicate where the promoter has been edited. Upper bars show the wild type genotype of the region being edited. Lower bars show the same region containing whatever edits, were made which are indicated by capital letters. The wild type does not have bars, as it is unedited. Bases written in a colored font are provided to indicate bases which are part of the binding site depicted in that color in Figure 1A. Panels depicting a FACS histogram were created using 10,000 cells of the strain represented by the lower bar. Strain data error bars indicate SEM (n=2). A. GAL1 expression from the strain shown in Figure 2B. (strain GE118) B. GAL1 expression from the strain shown in Figure 2C (strain GE168).

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9

Figure S7. Detailed investigation of the interior base pair contribution to the activity of the third Gal4 binding site in the GAL1 promoter. Related to Figure 4E. Black arrows indicate where the promoter has been edited. Upper bars show the wild type genotype of the region being edited. Upper bars show the wild type genotype of the region being edited. Lower bars show the same region containing whatever edits, were made which are indicated by capital letters. The wild type does not have bars, as it is unedited. Bases written in a colored font are provided to indicate bases which are part of the binding site depicted in that color in Figure 1A. Panels depicting a FACS histogram were created using 10,000 cells of the strain represented by the lower bar. Strain data error bars indicate SEM (n=2). A. Expression histogram from an edited GAL1 promoter (with edits such that the first two base pairs of the third Gal4 binding site interior are recoded; strain GE185), resulting in wild type expression. B. Expression histogram from an edited GAL1 promoter (with edits such that bases 3-5 of the third Gal4 binding site interior are recoded; strain GE186), resulting in a loss of expression. C. Expression histogram from an edited GAL1 promoter (with edits such that bases 6-8 of the third Gal4 binding site interior are recoded; strain GE187), resulting in a loss of expression. D. Expression histogram from an edited GAL1 promoter (with edits such that bases 9-11 of the third Gal4 binding site interior are recoded; strain GE188), resulting in wild type expression of GAL1 for a strain in which bases 9-11 of the third Gal4 binding site interior are edited. E. Expression histogram from an edited GAL1 promoter (with the fourth base of the third Gal4 binding site interior recoded; strain GE207), resulting in a loss of expression. F. Expression histogram from an edited GAL1 promoter (with the fifth base of the third Gal4 binding site interior recoded; strain GE208), resulting in wild type. G. Expression histogram from an edited GAL1 promoter (with the sixth base of the third Gal4 binding site interior recoded; strain GE209), resulting in a loss of expression.

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Figure S8. Detailed investigation of the interior base pair contribution to the activity of the third Gal4 binding site in the GAL10 promoter. Related to Figure 4E. Black arrows indicate where the promoter has been edited. Upper bars show the wild type genotype of the region being edited. Upper bars show the wild type genotype of the region being edited. Lower bars show the same region containing whatever edits, were made which are indicated by capital letters. The wild type does not have bars, as it is unedited. Bases written in a colored font are provided to indicate bases which are part of the binding site depicted in that color in Figure 1A. Panels depicting a FACS histogram were created using 10,000 cells of the strain represented by the lower bar. Strain data error bars indicate SEM (n=2). A. Expression histogram from an edited GAL10 promoter (with edits such that the first two base pairs of the third Gal4 binding site interior are recoded; strain GE200), resulting in a loss of expression. B. Expression histogram from an edited GAL10 promoter (with edits such that bases 3-5 of the third Gal4 binding site interior are recoded; strain GE201), resulting in a loss of expression. C. Expression histogram from an edited GAL10 promoter (with edits such that bases 6-8 of the third Gal4 binding site interior are recoded; strain GE202), resulting in a loss of expression. D. Expression histogram from an edited GAL10 promoter (with edits such that bases 9-11 of the third Gal4 binding site interior are recoded; strain GE203), resulting in a loss of expression. E. Expression histogram from an edited GAL10 promoter (with the fourth base of the third Gal4 binding site interior recoded; strain GE215), resulting in a loss of expression. F. Expression histogram from an edited GAL10 promoter (with the fifth base of the third Gal4 binding site interior recoded; strain GE216), resulting in wild type expression. G. Expression histogram from an edited GAL10 promoter (with the sixth base of the third Gal4 binding site interior recoded; strain GE217), resulting in wild type expression.

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Figure S9. Galactose concentration does not influence the behavior of edited strains. Related to Figure 6. Black arrows indicate where the promoter has been edited. Upper bars show the wild type genotype of the region being edited. Upper bars show the wild type genotype of the region being edited. Lower bars show the same region containing whatever edits, were made which are indicated by capital letters. The wild type does not have bars, as it is unedited. Bases written in a colored font are provided to indicate bases which are part of the binding site depicted in that color in Figure 1A. Percentages were generated from FACS histograms created using 10,000 cells of the strain represented by the lower bar. Strain data error bars indicate SEM (n=2). All growths were conducted in 0.1% mannose with either 0.175% (blue), 0.5% (red), or 2.0% (green) galactose included. A. Comparison of wild type promoter expression to an edited strain with the three base pairs immediately upstream of the first Gal4 binding site for both GAL1 (strains GE1, GE210) and GAL10 (strains GE105, GE218). B. Comparison of wild type promoter expression to an edited strain with the four base pairs 6bp downstream of the fourth Gal4 binding site for both GAL1 (strains GE1, GE151) and GAL10 (strains GE105, GE168). C. Comparison of wild type promoter expression to an edited strain with three base pairs in the interior of the third Gal4 binding site recoded for both GAL1 (strains GE1, GE186) and GAL10 (strains GE105, GE201). D. Comparison of wild type promoter (strains GE1) expression to two edited strains with 11bp between the third and fourth Gal4 binding sites either deleted (Edit 1; strain GE155) or recoded (Edit 2; strain GE206) for GAL1.

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II.

Supplemental Tables and Legends

Strain Name

Genetic Description

WP35

Matα, ho::HIS5-PGAL1WT-YFP

GE1

GE105

Matα, ho::HIS5-PGAL1WT-YFP, trp1::TRP1-PTEF1-CAS9 Matα, ho::HIS5-PGAL1WT-YFP, trp1::TRP1-PTEF1-CAS9, GAL::GAL10-PGAL1/106-GAL1 Matα, ho::HIS5-PGAL10WT-YFP, trp1::TRP1-PTEF1-CAS9

GE117

Matα, ho::HIS5-PGAL1117-YFP, trp1::TRP1-PTEF1-CAS9

GE118

GE135

Matα, ho::HIS5-PGAL1118-YFP, trp1::TRP1-PTEF1-CAS9 Matα, ho::HIS5-PGAL1WT-YFP, trp1::TRP1-PTEF1-CAS9, GAL::GAL10-PGAL1/10123-GAL1 Matα, ho::HIS5-PGAL1WT-YFP, trp1::TRP1-PTEF1-CAS9, GAL::GAL10-PGAL1/10124-GAL1 Matα, ho::HIS5-PGAL10135-YFP, trp1::TRP1-PTEF1-CAS9

GE140


GE149


GE151


GE153


GE155


GE156


GE163


GE164


GE165


GE166


GE168


GE170


GE182


GE183


GE184


GE185


GE186


GE187


GE188


GE190


GE192


GE193


GE6

GE123 GE124

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GE194


GE 195


GE196


GE197


GE200


GE201


GE202


GE203


GE204


GE 206


GE 207


GE 208


GE 209


GE 210


GE 215


GE 216


GE 217


GE 218

Matα, ho::HIS5-PGAL10218-YFP, trp1::TRP1-PTEF1-CAS9 Matα, ho::GAL10-PGAL1/10WT-GAL1, trp1::TRP1-PTEF1-CAS9, gal:: GAL10Truncated-PGAL1229-YFP Matα, ho::HIS5-PGAL1WT-YFP, trp1::TRP1-PTEF1-CAS9, GAL::KanMX

GE 229 GE 230 GE 231 GE 234 GE 235 GE 236 GE 241

Matα, ho::HIS5-PGAL1186-YFP, trp1::TRP1-PTEF1-CAS9, GAL::KanMX Matα, ho::GAL10-PGAL1/10WT-GAL1, trp1::TRP1-PTEF1-CAS9, gal:: GAL10Truncated-PGAL1WT-YFP Matα, ho::GAL10-PGAL1/10WT-GAL1, trp1::TRP1-PTEF1-CAS9, gal:: GAL10Truncated-PGAL1210-YFP Matα, ho::GAL10-PGAL1/10WT-GAL1, trp1::TRP1-PTEF1-CAS9, gal:: GAL10Truncated-PGAL10218-YFP Matα, ho::HIS5-PGAL1241-YFP, trp1::TRP1-PTEF1-CAS9

Table S1: Yeast strains constructed. Related to Figures 1-6. All strains originate from an unedited W303 strain and contain the edits shown above. The superscript number following a promoter indicates that the promoter has been edited and references the number of the specific edited strain which can be found below. ‘WT’ indicates that the promoter has not been edited although it has been placed in a new location and is driving YFP.

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Strain

5’ Region

UAS

3’ Region

S. cerevisiae

TATATTGAAGTA

UAS

AACAATAAAGATTC

S. paradoxus

AATATTGAAGTA

UAS

AACAATAAGGATTC

S. mikatae

-ATATTGAAATA

UAS

AACTATAATACTGG

S. bayanus

AATGTTGAAATA

UAS

AACAATGCAAATGC

Table S2: Comparison of the flanking regions of the GAL1/GAL10 upstream activation region (UAS) in three related yeast species. Related to Figure 2. The first column indicates the strain in question. The second column depicts the 12bp immediately 5’ of the UAS region defined as the region beginning with the first Gal4 binding site and ending with the last Gal4 binding site. The third column is a space holder where the 118 bp UAS resides. The last column depicts the 14bp immediately 3’ of the UAS region. Note that in S. mikatae the first base depicted is not present.

Binding Site

Beginning

Middle

End

GAL1/GAL10 #1

CGG

ATTAGAAGCCG

CCG

GAL1/GAL10 #2

CGG

GCGACAGCCCT

CCG

GAL1/GAL10 #3

CGG

AAGACTCTCCT

CCG

GAL1/GAL10 #4

CGC

GCCGCACTGCT

CCG

GAL80

CGG

CGCACTCTCGC

CCG

GAL3

CGG

TCCACTGTGTC

CCG

Table S3: Comparison of Gal4 binding site sequences from several locations within S. cerevisiae. Related to Figure 4. The first column describes the location of the binding site in question. The second column represents the first 3bp of the binding site which is canonically necessary for Gal4 binding. The third column depicts the middle 11bp of the binding site which has historically been considered irrelevant for Gal4 binding. The last column represents the last 3bp of the binding site which is also canonically necessary for Gal4 binding.

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